Gene expression in eukaryotes is often under the control of multiple nuclear proteins that work in combination to regulate transcription. The combined interactions between these proteins often create a new form of regulatory activity that is distinct from either protein alone. The interactions between proteins dictate which DNA target sites are bound and therefore which sets of genes are regulated. In some cases, these protein interactions also influence whether a regulatory protein functions as a transcriptional activator or repressor. This form of regulation, often referred to as the combinatorial control of transcription, is widely used to control gene expression under specific cellular or developmental conditions. Understanding the mechanism of how these proteins interact in different combinations, and how these interactions determine their activity and specificity of the complex will provide insight into the regulation of many developmental and cellular processes. We have chosen two regulatory systems in the yeast Saccharomyces cerevisiae to investigate the mechanism of combinatorial control of transcription. The alpha2 repressor, a homeodomain protein, interacts with the Mcm1 and a1 protein to turn off two different sets of cell type specific-genes in yeast. We have previously investigated how alpha2 recognizes the DNA on its own, and how the interactions with Mcml and a1 influence the target specificity of the complex. We now propose to investigate how the a1-alpha2 complex binding to multiple sites in the HO promoter function to repress transcription of the gene (Specific aim 1). The HO gene contains a large and complex promoter with a wide array of different regulatory sites and serves as a good model system for the regulation of developmental genes in higher eukaryotes. The yeast Mcm1 protein is a transcriptional regulatory factor with sequence similarity to the MADS-box DNA-binding domains of the mammalian serum response factor (SRF) and Myocyte Enhancer Factor 2A (MEF2A) proteins. The MADS-box domain of Mcm1 interacts with at least five different cofactors to regulate different sets of genes that are required for cellular processes ranging from cell mating type and arginine metabolism to cell-cycle control. We propose to investigate how Mcm1 interacts with the MATalpha1 protein and how this interaction alters the conformation of the protein to activate transcription (Specific aim 2). We propose to investigate the interactions of Mcm1 with SFF, a complex of proteins including the forkhead protein, Fkh2, that regulate cell-cycle specific genes (Specific aim 3). Finally, we plan to investigate the interactions of Mcm1 with ArgR, a complex of three proteins that regulate genes involved in arginine metabolism. The information we learn about the interactions between these different classes of proteins will be relevant to more complex systems in higher eukaryotes and will provide insight on how these proteins function to regulate development.